We are using a battery of biochemical techniques as well as recombinant DNA methods to determine how the large T-antigen of SV40 regulates gene expression and effects neoplastic transformation. Our studies indicate that large T-antigen interacts with the major groove of the SV40 DNA at three tandem binding sites in a sequential manner. More recently, we have shown that large T-antigen is an active enzyme capable of hydrolyzing ATP at a rate of 0.6 micromoles/hr/mg. The T-antigen ATPase is rapidly inhibited by monoclonal antibodies directed against large T and is greatly stimulated by poly d T. Moreover, homogeneously pure large T-antigen is able to bind specifically to a 54,000 dalton cellular phosphoprotein found in transformed cells but not detectable in normal cells. Finally, we have shown that small amounts of purified T-antigen can stimulate the production of sensitized lymphocytes capable of specifically killing SV40 transformed cells in vitro.